Viracept (nelfinavir mesylate, AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease

Feb 27, 2023 PARP

Viracept (nelfinavir mesylate, AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease. in cell culture. Comet-shaped foci Etravirine ( R165335, TMC125) occur upon convection-based transmission of cell-free viral particles from an infected cell to neighboring uninfected cells. HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shaped foci, indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensitive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and -D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growing round HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of nonlytic cell-to-cell transmission. Our study demonstrates the repurposing potential of nelfinavir with postexposure efficacy against different HAdVs and describes an alternative nonlytic cell-to-cell transmission mode of HAdV. (72,C74). The convection forces in the medium give rise to comet-shaped infection foci in cell cultures (72). Foci of infected cells are also found in tissue such as rat liver upon the intravenous inoculation of HAdV-C5 (75). Accordingly, acute HAdV infections trigger an inflammatory response, as shown in airways or conjunctiva of susceptible animals (2, 76). In contrast to lytic virus transmission, direct cell-to-cell transmission leads to round plaques, as shown with vaccinia virus (77,C80). The mechanisms of virus transmission are highly virus specific. They comprise nonlytic pathways involving secretory-endocytic circuits, multivesicular or autophagic membrane processes, cellular protrusions, or transient breaches of membrane integrity (80,C84). In contrast, lytic egress pathways further involve the destabilization of cellular membranes by viral and host factors, often tuned by the cytoskeleton (37, 85,C88). HAdV-C2 controls lytic cell death by the adenovirus death protein (ADP), also known as 11.6K, as concluded from genetic and IRF5 overexpression studies (73, 74). ADP is a type III membrane protein transcribed from the CR1- region in the immunoregulatory E3a locus. All HAdV-C members harbor homologous E3a CR1- sequences (e.g., 10.5K in HAdV-C5). Other HAdV species differ in their E3 regions, however (89,C91). The N terminus of ADP is luminal, and the C terminus protrudes into the cytosol (92). Following posttranslational modifications, ADP is transported to the inner nuclear membrane, where the N terminus is intruding into the nucleus (93). At late stages, when capsid assembly in the nucleus has commenced, Etravirine ( R165335, TMC125) ADP expression is boosted (94, 95). The mechanism of host cell lysis is still unknown, although necrosis-like, autophagic, and caspase activities have been implicated (96,C99). Here, we report that nelfinavir mesylate (nelfinavir for short) is an effective inhibitor of HAdV lytic egress. The procedure leading to the identification of nelfinavir is described in another study using an imaging-based, high-content screen of the Prestwick Chemical Library (PCL) comprising 1,280 mostly clinical or preclinical compounds (100, 101). Nelfinavir is the off-patent active pharmaceutical ingredient of Viracept, an FDA-approved drug that inhibits the human immunodeficiency virus (HIV) protease (102). The work here documents the repurposing potential of nelfinavir, which is effective against a spectrum of HAdV types in a postexposure manner. Nelfinavir is partly, but not exclusively, active against ADP-encoding HAdV types and uncovers the appearance of round plaques, which arise upon nonlytic cell-to-cell viral transmission. RESULTS Nelfinavir is a nontoxic, potent inhibitor of HAdV-C multicycle infection. A recent paper describes a full-cycle, image-based screen of 1 1,278 out of 1 1,280 PCL compounds against HAdV-C2-dE3B-GFP, where clopamide and amphotericin B were excluded due to precipitation during acoustic dispension into the screening plates (100). The screen was conducted in adenocarcinomic human alveolar basal epithelial (A549) cells at a 1.25?M compound concentration and identified Etravirine ( R165335, TMC125) nelfinavir, aminacrine, dequalinium dichloride, and thonzonium bromide as hits (see Table S1 in the supplemental material). Nelfinavir (CAS number 159989-65-8) strongly inhibited plaque formation at nanomolar concentrations, comparably to the known HAdV nucleoside analogue inhibitor 3-deoxy-3-fluorothymidine (DFT) (Fig. 1A and ?andB).B). Dequalinium dichloride, aminacrine, and thonzonium bromide were excluded from further analyses due to toxicity (100) and potential mutagenic effects (103). Long-term incubations of uninfected A549 cells with nelfinavir for up to 115 h showed median toxicity (concentration causing 50% toxicity [TC50]) values of 25.7?M, as determined by cell impedance measurements using xCELLigence (Fig. 1C). xCELLigence measures the impedance of electrical currents imposed by cell adherence to gold-plated microelectrodes implanted in culture wells. Impedance is expressed as a cell index (CI), a unitless parameter proportional to the Etravirine ( R165335, TMC125) cell number, cell size, and cell adherence. For raw CI profiles, see Fig. S1A in the supplemental material. CI measurements were consistent with.